Radio-immunoassays and radio-receptor assays using radiolabeled tracers are widely used for the specific quantitation of small amounts of antigens or receptors respectively in biological fluids. However, the sensitivity of these assays is limited by the specific activity of the radio-isotope; and, for example in studies of nicotinic acetylcholine (nACh) receptors in brain, this presents a problem due to the relatively low tissue concentration. Additionally, use of radioactive material presents serious problems in storage, handling and disposal.
Non-radioisotopic probes have been developed for use in immunoassays by conjugating antigens to compounds with fluorescent or chemiluminescent properties as well as to free radicals and enzymes. However, sensitivity of these immunoassays is generally not superior to the classical radioimmunoassays.
Bioluminescent immunoassays have been developed in which the ligand is covalently linked to firefly luciferase. Although the reported sensitivity of these bioluminescent assays is comparable to radioimmunoassays, and the theoretical sensitivity is greater due to the sensitivity of the luciferin-luciferase reaction, luciferase is somewhat unstable and is subject to degradation by proteolytic enzymes. Also, the luciferase molecule is relatively large and may introduce steric changes in the ligand to be studied.